1. Field Of the Invention
The invention concerns methods and compositions for the determination of peroxidatively active substances, assays involving the methods of the present invention, and kits for conducting such methods and assays.
Exemplary peroxidatively active substances are peroxidase enzymes. The peroxidases are a group of enzymes which catalyze the oxidation of specific substrates by hydroperoxides usually hydrogen peroxide. Certain substrates (chromogenie substrates), when oxidized in this way, become strong chromophores, generating intense color. The peroxidases have been widely used as labels in assays, usually as conjugates with specific binding pair (SBP) members as assay components. The presence of such a labelled component can be readily detected by adding a solution comprising the specific chromogenic substrate and a peroxide.
One of the more widely used peroxidases is horse radish peroxidase (HRP), for which a specific substrate is tetramethylbenzidine (TMB). HRP has found broad application as a marker enzyme in enzyme-immunological determination methods (enzyme immunoassays, EIA). A common feature of EIA systems is the exploitation of specific binding pair member interaction, such as an antigen-antibody interaction, for the determination of the presence or concentration of an analyte, e.g. antigen or antibody. A multitude of EIA systems have been described. These may be classified, for example, as systems with enzyme-labelled antigen and as systems with enzyme-labelled antibody.
One example of an EIA system with labelled antigen is the competitive solid phase EIA (enzyme linked immunosorbant assay, ELISA). In this system a known amount of an enzyme-labelled antigen and the antigen to be determined (analyte) compete for the limited number of binding sites of a specific antibody, which is immobilized on a solid phase. The higher the concentration of analyte, the lower the amount of enzyme-labelled antigen bound to the immobilized antibody. Making use of a calibration curve, the determination of the enzyme activity bound to the solid phase by interaction with the antibody allows the determination of the analyte concentration.
Another example is a labelled-antibody EIA which is a sandwich "assay" (or two site enzyme immunometric test). The assay includes two immunological reactions. In the first reaction a macromolecular antigen is bound more or less completely to an antibody immobilized on a solid phase. Next, the bound antigen is reacted with an excess of a conjugate consisting of a specific antibody and enzyme. The extent of the binding of the labelled antibody is proportional to the amount of antigen; thus, the determination of the remaining enzyme activity after removal of excess conjugate is a measure of the amount of antigen.
Another example of an EIA is a homogeneous enzyme immunoassay sold by Syva Company, Palo Alto, California under the trade name EMIT. In EMIT assays the reaction takes place in one step in solution. Typically, the analyte from the test sample is combined with an enzyme-labelled analyte analog. Both the analyte and enzyme-labelled analyte analog are capable of competing for binding sites on an antibody for the analyte which is included in the reaction medium. By comparison with known standards, the enzyme activity that remains after the sample and other reagents are combined is a measure of the amount of analyte in the sample.
Thus, the determination of enzyme activity is an essential step in all EIAs. In general, enzyme activity is determined by well-known procedures. Typically changes of optical properties (absorption, fluorescence, luminescence, and the like) of an appropriate substrate effected by the enzymatic action is followed. Appropriate measuring instruments are used, and a linear proportionality between measuring signal and an amount of enzyme is advantageous.
Numerous substrates for peroxidases, particularly horseradish peroxidase, have been reported. Many are chromogenic substrates that involve oxidation of a leuco dye to its colored form. Typical of this type of substrate is tetramethyl- benzidine, 3,3-bis-(3-carboxypropoxy)benzidine, N-ethyl-3-aminocarbazol, guiacol, azo-bis-tetramethyl-benzimidazole sulfonic acid (ABTS), etc. In another group of dye substrates, the oxidized substrate forms a color by reacting with its own unoxidized form. Examples of such self-coupling substrates include p-hydroxyphenylpropionic acid, 4-chloronaphthol, and o-phenylenediamine.
A second class of substrates, called "developers" are oxidized to intermediates that condense with color-forming molecules, called "couplers" to form dyes Typical of this class of substrates are N-methylbenzothiazolone hydrazone and aminoantipyrene, either of which on oxidation can couple to electrophilic compounds such as aminobenzoic acid, diketones, phenols, etc.
The second class of substrates are of special interest because HRP/H.sub.2 O.sub.2 catalyzes the condensation of two compounds. Ease of manipulation makes a dual substrate system attractive for various applications in EIA.
In certain applications a sensitive, precipitable, chromogenic substrate is used. One such substrate which has been recently published by a group at Kodak is precipitable and has the capability of detecting &lt;50 pg/M1 of HRP in 30 minutes (D. J. Dannet, et al., (1973) Arch Biochem Biophys., 156:759; P. E. Weller, et al. (1985) Arch Biochem. Biophys., 243(2):633-643; European Patent 02527457; H. B. Dunford et al. (1986) Arch Biochem Biophys, 251 (2):536-542; U.S. Pat. No. 4,089,747).
Benzidine and 3,3'-dimethoxybenzidine have been shown to oxidatively couple with 4-substituted or unsubstituted 2-t-butylphenols where the substituent is methoxy or halide (Josephy, etal.; Carcinogenesis (1982) 3 (10):1227-1230; and Biochem. Pharmacol. (1984) 33 (7):1155-1156). In these reactions the substituent, or hydrogen when there was no substituent, was displaced by the nitrogen of an oxidized benzidine to form a merocyanine dye. However, there was no recognition of the use of this reaction for determination of peroxidase activity or hydrogen peroxide concentration or of its use in assays for analytes. Further, there was no recognition of the use of the condensation reaction for generating a detectable signal by cleaving a bond to a signal suppressing substituent as where the substrates are of the formula S-L-M, wherein S is a signal generating moiety whose signal is modulated by signal modulating moiety M when M is bound to S, and L is a bond, or a linking group having a bond, which is capable of being cleaved by the reaction of PAS with a substrate of PAS and a hydrogen donor.
Also related to this class of substrates are dye substrates such as p-hydroxy-and p-amino-, particularly p-dialkylamino-, anilines. Some dye substrates of this class, after oxidization, condense with couplers and undergo bond cleavage resulting in the release of a second dye molecule and a molecule capable of fluorescing. In these cases the coupler was a p-amino- or p-hydroxy-phenyl ether of a dye molecule, and the substrates were used to determine hydrogen peroxide concentrations. Examples of this type of substrate are disclosed in European patent specification, publication No. 0060518 B1 (EPO patent application 82101947.8). Again there was no recognition of whether or not these compounds were even useful for the determination of peroxidase activity; or of the use of the condensation reaction for generating a detectable signal by cleaving a bond to a signal suppressing substituent as where the substrates are of the formula S-L-M, wherein S is a signal generating moiety whose signal is modulated by signal modulating moiety M when M is bound to S, and L is a bond, or a linking group having a bond, which is capable of being cleaved by the reaction of PAS with a substrate of PAS and a hydrogen donor. On the other hand, the compositions of the present invention are useful for the determination of peroxidase activity and are comprised of a structural moiety that is capable of producing a signal and a structural moiety that modulates the signal produced by the signal producing moiety. An example of the present compositions are compounds comprised of a signal group capable of fluorescing linked to a signal modulating group capable of quenching the fluorescence of the signal group by energy transfer.
2. Description of the Related Art
European patent specification, publication No. 0060518 B1, as set forth above, discloses a reagent for assaying hydrogen peroxide and a method of quantitative assay for hydrogen peroxide.
Latt, S.A.; et al. in Anal. Biochem. (1972) 50 (1):56-62 disclose a fluorescence determination of carboxypeptidase A using a poly(amino acid) substrate analog having two fluorescent labels, one of which is quenched when bound to the substrate analog. Upon hydrolysis of the quenched label by carboxypeptidase A, its fluorescence is restored. The reaction is said to be useful for mechanistic research, assays for inhibition of cobalt carboxypeptidase by amino acids (e.g. L-phenylalanine) and assays for peptidase activity.
Josephy, al. in Carcinoaenesis (1982) 3 (10):1227-1230 discloses the chemical structures of adducts formed by the oxidation of benzidine in the presence of phenols. Josephy, et al. in Biochem. Pharmacol. (1984) 33 (7):1155-1156 discloses thereaction of 4-substituted phenols with benzidine in a peroxidase system.
U.S. Pat. No. 4,853,328 discloses a reagent for assaying hydrogen peroxide and a method of quantitative assay for hydrogen peroxide.
PCT International Application, Publication No. WO86/05207, discusses improvements relating to assay reagents, which are peroxidase-containing and tetramethylbenzidine peroxidase-substrate reagents.
European Patent Application 0123902 A2 discloses a procedure for the determination of peroxidase activity by end dilution titration and means for its realization. The procedure is carried out with a benzidine derivative in the presence of a non-chromogenic or low-chromogenic phenylene derivative, which is a proton donor, to retard color development for a sufficient period of time to permit a sharp dilution end point and thereafter detecting the peroxidase activity.
U.S. patent application No. 4,279,993 discloses an indicator composition and test device containing amine oxide and a method of its use. The composition contains a benzidine-type indicator.
Semi-quantitative determination of urine glucose with a carrier matrix containing glucose oxidase, a peroxidase and m-anisidine, optionally with tetramethyl- benzidine is disclosed in U.S. Pat. No. 4,391,905 and 4,391,906.
U.S. Pat. No. 4,556,640 discusses a stabilized test composition, device and method for the determination of peroxidatively active substances.
Ionic compounds containing the cationic meriquinone of a benzidine are disclosed in U.S. Pat. No. 4,789,630.
European Patent Application, Publication No. 0060518 A1 discusses reagents for assay of hydrogen peroxide especially in diagnostic systems with a chemical moiety and fluorescing moiety present.
A test composition for peroxidatively active substance with aniline derivative present as a stabilizer and device and method for the determination of peroxidatively active substances is disclosed in European Patent Application, Publication No. 0130520 A1.
Ethanol determination in aqueous samples with alcohol oxidase, peroxidase, and a reduced chromogenic indicator is disclosed in European Patent Application, Publication No. 0164008 A2.
U.S. Pat. No. 4,587,220 discusses ascorbate interference-resistant composition, device and method for the determination of peroxidatively active substances.
Quantitative determination of hemoglobin and cytochemicals staining for peroxidase using 3,3'-5,5'-tetramethylbenzidine dihydrochloride as a safe substitute for benzidine is disclosed by H. H. Liem, et al. (1979) Analytical Biochemistry 98:388-393.
Europaische Patentanmeldung 0224210 A1 discloses a color forming reagent for measuring peroxidase activity containing tetralkylbenzidine, peroxide and acidic buffer to improve sensitivity.
Stabilized enzyme substrate solutions are disclosed in PCT International Application, Publication No. WO 86/04610.
U.S. Pat. No. 4,503,143 discloses an enzyme immunoassay with two-parts solution of tetramethylbenzidine as chromogen.
A test composition for the detection of peroxidatively active substances is disclosed in UK Patent Specification 1560077.
U.S. Pat. No. 3,527,331 discusses substantially anhydrous, solid assay materials for the determination of reagent for assaying aldolase. The materials are rendered storage stable by the presence of certain polyhydric compounds preferably mannitol, sorbitol, lactose or polyvinyl alcohol. An indicator composition and test device containing amine oxide and a method of its use is disclosed in U.S. Pat. No. 4,279,993.
U.S. Pat. No. 4,310,626 discloses interference-resistent composition, device and method for determining a peroxidatively active substance in a test sample.
Stabilization of indicators for detecting enzyme activity is disclosed in U.S. Pat. No. 4,615,972.
An assay for peroxidative enzyme activity is discussed in U.S. Pat. No. 4,596,770.
A specific binding assay employing an enzyme-cleavable substrate as a label is disclosed in U.S. Pat. No. 4,279,992.
An agent for the determination of peroxidase activity, with stabilizer, is disclosed in U.S. Pat. No. 4,891,314.